Organ and tissue generation from stem cells and their successive transplantation provide possible treatments for a number of pathologies, making stem cells a central focus of research in many fields. Using stem cells for generation of organs and tissues for transplantation provides a possible therapy for diabetes, Parkinson's disease, liver disease, heart disease, and autoimmune disorders, to name a few. However, there are at least two major problems associated with organ and tissue transplantation. First, there is a shortage of donor organs and tissues. As few as 10 percent of the organs needed for transplant in Italy along ever become available to a recipient. See, e.g., Nord Italian Transplant program report 2007. According to the Nord Italian Transplant program report, only about 1,200 of the 9,000 Italians who needed a new kidney in 2006 received one, and that in 2006 an average of 12% of the patients in the waiting list for a liver transplant dic while waiting to receive the suitable organ. The second major problem is the potential incompatibility of the transplanted tissue with the immune system of the recipient. Because the donated organ or tissue is recognized by the host immune system as foreign, anti-rejection medications must be provided to the patient at a significant cost, both financially and physically.
Xenotransplantation, or transplantation of tissue or organs from another species, could provide an alternative possibility to overcome the shortage of human organs and tissues. Xenotransplantation would offer the advantage of advanced planning of the transplant, allowing the organ to be harvested while still healthy and allowing the patient to undergo any possible pre-treatment prior to transplant surgery. Unfortunately, xenotransplantation does not overcome the problem of tissue incompatibility, but even exacerbates it. Furthermore, according to the Centers for Disease Control, there is evidence that damaging viruses cross species barriers. Pigs have become likely candidates as organ and tissue donors, yet cross-species transmission of more than one virus from pigs to humans has been documented. For example, over a million pigs were reported slaughtered in Malaysia in an effort to contain an outbreak of Hendra virus, a disease that was transmitted to more than 70 humans with deadly results. See, e.g., Butler, D., Nature (1999) 398: 549.
A promising source of organs and tissues for transplantation therefore lies in the development of stem cell technology. Theoretically, stem cells can undergo self-renewing cell division to give rise to phenotypically and genotypically identical daughters for an indefinite time and ultimately can differentiate into at least one final cell type. By generating tissues or organs from a patient's own stem cells, or by genetically altering heterologous cells so that the recipient immune system does not recognize them as foreign, transplant tissues can be generated to provide the advantages associated with xenotransplantation without the associated risk of infection or tissue rejection.
Stem cells also provide promise for improving the results of gene therapy. A patient's own stem cells could be genetically altered in vitro, then reintroduced in vivo to produce a desired gene product. These genetically altered stem cells would have the potential to be induced to differentiate to form a multitude of cell types for implantation at specific sites in the body, or for systemic application. Alternatively, heterologous stem cells could be genetically altered to express the recipient's major histocompatibility complex (MHC) antigen, or no MHC, to allow transplant of those cells from donor to recipient without the associated risk of rejection.
Stem cells are cells that have extensive, possibly indefinite, proliferation potential to differentiate into several cell lineages and can repopulate tissues upon transplantation. The quintessential stem cell is the embryonic stem (ES) cell, as it has unlimited self-renewal and multipotent differentiation potential. These cells are derived from the inner cell mass of the blastocyst, or can be derived from the primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived from mouse, and more recently also from non-human primates and humans. When introduced into mouse blastocysts or blastocysts of other animals, ES cells can contribute to all tissues of the mouse (animal). When transplanted in post-natal animals, ES and EG cells generate teratomas, which again demonstrates their multipotency. ES (and EG) cells can be identified by positive staining with the antibodies SSEA-1 and SSEA-4.
At the molecular level, ES and EG cells express a number of transcription factors highly specific for these undifferentiated cells. These include Oct-4 and Nanog. Also found are the LIF-R and the transcription factors Sox-2 and Rox-1, even though the latter two are also expressed in non-ES cells. Oct-4 is a transcription factor expressed in the pre-gastrulation embryo, early cleavage stage embryo, cells of the inner cell mass of the blastocyst, and in embryonic carcinoma (EC) cells. Oct-4 is down-regulated when cells are induced to differentiate in vitro and in the adult animal. Oct-4 is only found in germ cells. Several studies have shown that Oct-4 is required for maintaining the undifferentiated phenotype of ES cells and plays a major role in determining early steps in embryogenesis and differentiation. Oct-4, in combination with Rox-1, causes transcriptional activation of the Zn-finger protein Rex-1 and is also required for maintaining ES in an undifferentiated state. Human or murine primordial germ cells require presence of LIF. Another hallmark of ES cells is presence of telomerase, which provides these cells with an unlimited self-renewal potential in vitro.
Stem cells have been identified in most organ tissues. The best characterized is the hematopoietic stem cell. This is a mesoderm-derived cell that has been purified based on cell surface markers and functional characteristics. The hematopoietic stem cell, isolated from bone marrow, blood, cord blood, fetal liver and yolk sac, is the progenitor cell that reinitiates hematopoiesis for the life of a recipient and generates multiple hematopoietic lineages (see Fei, R., et al., U.S. Pat. No. 5,635,387; McGlave, et al., U.S. Pat. No. 5,460,964; Simmons, P., et al., U.S. Pat. No. 5,677,136; Tsukamoto, et al., U.S. Pat. No. 5,750,397; Schwartz, et al., U.S. Pat. No. 5,759,793; DiGuisto, et al., U.S. Pat. No. 5,681,599; Tsukamoto, et al., U.S. Pat. No. 5,716,827; Hill, B., et al., Exp. Hematol. (1996) 24(8): 936-943). When transplanted into lethally irradiated animals or humans, hematopoietic stem cells can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hemopoietic cell pool. In vitro, hemopoietic stem cells can be induced to undergo at least some self-renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. Therefore, this cell fulfills the criteria of a stein cell. Stem cells which differentiate only to form cells of hematopoietic lineage, however, are unable to provide a source of cells for repair of other damaged tissues, for example, heart or lung tissue damaged by high-dose chemotherapeutic agents.
A second stem cell that has been studied extensively is the neural stem cell (Gage F. H., Science 287:1433-1438, 2000); Svendsen C. N., et al, Brain Path. 9:499-513, 1999; Okabe S., et al, Mech. Dev. 59:89-102, 1996). Neural stem cells were initially identified in the subventricular zone and the olfactory bulb of fetal brain. Until recently, it was believed that the adult brain no longer contained cells with stem cell potential. However, several studies in rodents, and more recently also non-human primates and humans, have shown that stem cells continue to be present in adult brain. These stem cells can proliferate in vivo and continuously regenerate at least some neuronal cells in vivo. When cultured ex vivo, neural stem cells can be induced to proliferate, as well as to differentiate into different types of neurons and glial cells. When transplanted into the brain, neural stem cells can engraft and generate neural cells and glial cells. Therefore, this cell falls within the scope of a stem cell.
Mesenchymal stem cells (MSC), originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. During embryogenesis, the mesoderm develops into limb-bud mesoderm, tissue that generates bone, cartilage, fat, skeletal muscle and possibly endothelium. Mesoderm also differentiates to visceral mesoderm, which can give rise to cardiac muscle, smooth muscle, or blood islands consisting of endothelium and hematopoietic progenitor cells. Primitive mesodermal or mesenchymal stem cells, therefore, could provide a source for a number of cell and tissue types. A third tissue specific cell that has been named a stem cell is the mesenchymal stem cell, initially described by Fridenshtein (Fridenshtein, Arkh. Patol., 44:3-11, 1982). A number of mesenchymal stem cells have been isolated (see, for example, Caplan, A., et al., U.S. Pat. No. 5,486,359; Young. H., et al., U.S. Pat. No. 5,827,735; Caplan, A., et al., U.S. Pat. No. 5,811,094; Bruder, S., et al., U.S. Pat. No. 5,736,396; Caplan, A., et al., U.S. Pat. No. 5,837,539; Masinovsky, B., U.S. Pat. No. 5,837,670; Pittenger, M., U.S. Pat. No. 5,827,740; Jaiswal, N., et al., J. Cell Biochem. (1997) 64(2): 295-312; Cassiede P., et al., J. Bone Miner. Res. (1996) 11(9): 1264-1273; Johnstone, B., et al., Exp. Cell Res. (1998) 238(1): 265-272; Yoo, et al., J. Bone Joint Sure. Am. (1998) 80(12): 1745-1757; Gronthos, S., Blood (1994) 84(12): 4164-4173; Makino, S., et al., J. Clin. Invest. (1999) 103(5): 697-705). Of the many mesenchymal stem cells that have been described, all have demonstrated limited differentiation to form only those differentiated cells generally considered to be of mesenchymal origin. To date, the most multipotent mesenchymal stem cell reported is the cell isolated by Pittenger, et al., which expresses the SH2+ SH4+ CD29+ CD44+ CD71+ CD90+ CD106+ CD120a+CD124+CD14− CD34− CD45− phenotype. This cell is capable of differentiating to form a number of cell types of mesenchymal origin, but has been reported by the team who isolated it to be apparently limited in differentiation potential to cells of the mesenchymal lineage since hematopoietic cells were never identified in the expanded cultures. (Pittenger, et al., Science (1999) 284: 143-147.)
Other stem cells have been identified, including gastrointestinal stem cells, epidermal stem cells, and hepatic stem cells, also termed oval cells (Potten C., Philos Trans R Soc Lond B Biol Sci 353:821-30, 1998; Watt F., Philos. Trans R Soc Lond B Biol Sci 353:831, 1997; Alison M., et al, Hepatol 29:678-83, 1998).
Compared with ES cells, tissue specific stem cells have less self-renewal ability and, although they differentiate into multiple lineages, they are not as pluripotent. In addition, the degree of telomerase activity in tissue specific stem cells has not been fully explored, in part because large numbers of highly enriched populations of these cells are difficult to obtain.
Until recently, it was thought that organ specific stem cells could only differentiate into cells of the same tissue. A number of recent publications have suggested that adult organ specific stem cells may be capable of differentiating into cells of different tissues. A number of studies have shown that cells transplanted at the time of a bone marrow transplant can differentiate into skeletal muscle (Ferrari, Science 279:528-30, 1998; Gussoni, Nature 401:390-4, 1999). This could be considered within the realm of possible differentiation potential of mesenchymal cells that are present in marrow. Jackson reported that muscle satellite cells can differentiate into hemopoietic cells, again a switch in phenotype within the splanchnic mesoderm (Jackson, PNAS USA, 96:14482-6, 1999). Other studies have shown that stem cells from one embryonal layer (e.g., splanchnic mesoderm) can differentiate into tissues thought to be derived during embryogenesis from a different embryonal layer. For example, endothelial cells or their precursors detected in humans or animals that underwent marrow transplantation are at least in part derived from the marrow donor (Takahashi, Nat Med 5:434-8, 1999; Lin, Clin Invest 105:71-7, 2000). Thus, visceral mesoderm and not splanchnic mesoderm, such as MSC, derived progeny are transferred with the infused marrow. Even more surprising are the reports demonstrating both in rodents and humans that hepatic epithelial cells and biliary duct epithelial cells are derived from the donor marrow (Petersen, Science 284:1168-1170, 1999; Theise, Hepatology 31:235-40, 2000; Theise, Hepatology 32:11-6, 2000). Likewise, three groups have shown that neural stem cells can differentiate into hemopoietic cells. Finally, Clarke et al. reported that neural stem cells injected into blastocysts can contribute to all tissues of the chimeric mouse (Clarke, Science 288:1660-3, 2000).
Many of these studies have not conclusively demonstrated that a single cell can differentiate into tissues of different organs. Many investigators did not identify the phenotype of the initiating cell. An exception is the study by Weissman and Grompe, who showed that cells that repopulated the liver were present in Lin−ThytLowSca1+ marrow cells, which are highly enriched in hematopoietic stem cells. Likewise, the Mulligan group showed that marrow Sp cells, highly enriched for HSC, can differentiate into muscle and endothelium, and Jackson et al. showed that muscle Sp cells are responsible for hemopoietic reconstitution (Gussoni et al., Nature 401:390-4, 1999).
Transplantation of tissues and organs generated from heterologous embryonic stem cells requires either that the cells be further genetically modified to inhibit expression of certain cell surface markers, or that the use of chemotherapeutic immune suppressors continue in order to protect against transplant rejection. Thus, although embryonic stem cell research provides a promising alternative solution to the problem of a limited supply of organs for transplantation, the problems and risks associated with the need for immunosuppression to sustain transplantation of heterologous cells or tissue would remain. An estimated 20 immunologically different lines of embryonic stem cells would need to be established in order to provide immunocompatible cells for therapies directed to the majority of the population (Wadman, M., Nature (1999) 398: 551).
Using cells from the developed individual, rather than an embryo, as a source of autologous or allogeneic stem cells would overcome the problem of tissue incompatibility associated with the use of transplanted embryonic stem cells, as well as solve the ethical dilemma associated with embryonic stem cell research. The greatest disadvantage associated with the use of autologous stem cells for tissue transplant thus far lies in their limited differentiation potential. A number of stem cells have been isolated from fully-developed organisms, particularly humans, but these cells, although reported to be pluripotent, have demonstrated limited potential to differentiate to multiple cell types.
Thus, even though stem cells with multiple differentiation potential have been isolated previously by others and by the present inventors, a progenitor cell with the potential to differentiate into a wide variety of cell types of different lineages, including fibroblasts, osteoblasts, chondrocytes, adipocytes, skeletal muscle, endothelium, stroma, smooth muscle, cardiac muscle and hemapoietic cells, has not been described. If cell and tissue transplant and gene therapy are to provide the therapeutic advances expected, a stem cell or progenitor cell with the greatest or most extensive differentiation potential is needed. What is needed is the adult equivalent of an embryonic stem cell.
As an alternative to embryonic stem cell therapy, adult stem cells have shown promise (Caplan, 1991, 2000, 2003, 2004, 2005; Caplan and Bruder, 2001; Kuehle and Goodell, 2002; Pittenger, 2004). For example, multipotent adult progenitor cells from mouse bone marrow (mMAPC) were shown to express several embryonic stem (ES) cell markers, such as Oct-4 (POU transcription factor), Rex-1 (transcription factor) and SSEA-1 (stage-specific embryonic antigen), and to contribute to all embryonic cell lineages when a single cell is injected into the blastocyst (Jiang et al., 2002). While bone marrow is an excellent source of stem cells with proven therapeutic value, the process of collecting bone marrow is invasive, and, moreover, recent data implicate bone marrow stem cells in cancer development (Houghton et al., 2004). The expansion of the list of the potential sources of pluripotent adult stem cells beyond a small group consisting of cord blood, bone marrow, adipose tissue, and amniotic stem cells (Jiang et al., 2002; Zuk et al., 2002; Miki et al., 2005) would be of value. Extending earlier findings in rodents (Mann et al., 1996), the recent discovery of relatively immature stem cells in the dental pulp of human exfoliated deciduous teeth (SHED) has offered a potentially non-invasive source of stem cells (Miura et al., 2003). SHED showed rapid expansion and proliferation in vitro while expressing several mesenchymal stem cell markers, such as STRO-1 and CD146. Stem cells from dental pulp (Miura et al., 2003) appeared to be inferior in their potential therapeutic value compared to ES cells or mMAPCs, since they were not shown to express Oct-4, SSEAs, Nanog, or any other hallmarks of totipotent stem cells, while their multilineage terminal differentiation was only marginally successful (Jiang et al., 2002; Chambers et al., 2003; Constantinescu, 2003; Laslett et al., 2003; Mitsui et al., 2003; Pierdomenico et al., 2005; Laino et al., 2006). SHED have been shown to be highly heterogeneous, because only 9% of SHED express markers of undifferentiated cells, and it is not clear if clones obtained from SHED maintain expression of these markers (Miura et al., 2003). Previously, it has been reported that removal of stem cells from their natural milieu may change their differentiation properties (Bissell and Lafarge, 2005; Schwartz and Verfaillie, 2005). Additional publications that described stem cells obtained from dental pulp include: WO 03/066840, WO 04/094588, US 2005/0106724, US 2007/0009492, US 2007/0258957, WO 03/066840, EP 1748066 A1, WO 2006/010600, and WO 2006/100088. However, none of these references describe a homogeneous population of purified stem cells which can differentiate into cells of different lineages.
Accordingly, what is needed is a population of stem cells which show the plasticity of ES cells in their ability to become a multitude of cells but derived from non-embryonic sources and non-invasive sources. The present invention fulfils these needs and provides additional benefits as well.
The specification is most thoroughly understood in light of the references cited herein. Some of the references have their full bibliographic information after the Examples section. The disclosures of all publications, patents, patent applications, and published patent applications referred to herein are each hereby incorporated by reference herein in their entireties.